Soybean (Glycine max) is an important economic crop. Herbicide (especially glyphosate) tolerance is one of the critical traits for desirable soybean. Glyphosate, a kind of herbicide that has activity on broad spectra of plant species, is the major ingredient of Roundup® (Monsanto Colo., St. Louis, Mo.), a safe herbicide with short half-life in environment. When applied to the surfaces of the plants, glyphosate moves systemically through the entire plants. Glyphosate is phytotoxic because of its inhibition of the shikimic acid pathway, which provides a precursor for the synthesis of aromatic amino acids. Glyphosate inhibits the enolpyruvyl-3-phosphoshikimate synthase (EPSPS) found in plants.
Glyphosate tolerance may be obtained by expressinG mutants that have lower affinity for glyphosate and retain metabolic activity in the presence of glyphosate (U.S. Pat. Nos. 5,633,435; 5,094,945; 4,535,060 and 6,040,497). Glyphosate degrading enzymes in plant tissues (U.S. Pat. No. 5,463,175) are also capable of conferring glyphosate tolerance to cells. These genes are used for producing transgenic crops with tolerance to glyphosate, and consequently, glyphosate may be used for effective weed control with minimal damage to crops. Glyphosate tolerance has already been genetically engineered into corn (U.S. Pat. No. 5,554,798), wheat (U.S. Pat. No. 6,689,880), cotton (U.S. Pat. No. 6,740,488), soybean (WO 9200377), and canola (US Patent Appl. 20040018518) so far. The transgenes for glyphosate tolerance and the transgenes for other herbicides tolerance, e.g., the bar gene, (Toki el al., 1992; Thompson et al., 1987, for glufosinate herbicide tolerance) are also useful as selectable markers or scorable markers and may provide useful phenotypes for selection of plants in connection with other agronomically useful traits.
The expression of foreign genes in plants is influenced by their positions along chromosomes. Great differences are observed in transformants due to the different insertion sites of foreign genes. As a result, a large number of events need to be screened to identify the event characterized by the optimal expression of the introduced foreign gene. It has been observed in different transgenic events that there is a wide range of variation in the expression level of the introduced gene in different tissues. There are also different temporal and spatial expression patterns, such as the differences in the relative expression of a transgene in various plant tissues. Therefore, hundreds to thousands of different events need to be produced and screened for a single event that has the desired transgene expression levels and patterns for commercial purpose. An event with desired levels or patterns of transgene expression is helpful for introgressing the transgene into other genetic backgrounds through tradition methods of sexual crossing. The progeny thus obtained may maintain the transgene expression characteristics of the original transformant. The strategy ensures reliable expression in a number of varieties which are well adapted to local growing conditions.
The detection of the presence of a particular event may be accomplished by identifying whether a progeny of a sexual cross comprises the transgene of interest. Besides, the method for detecting a particular event is necessary to comply with regulations and requirements for the pre-market approval and labeling of foods derived from genetically modified crops. It is possible to detect the presence of a transgene by any nucleic acid examination method such as the polymerase chain reaction (PCR) or DNA hybridization using polynucleic acid probes. These detection methods are generally concerned with frequently used genetic elements, such as promoters, terminators, marker genes, etc., therefore, these methods are not valid for discriminating between different events, particularly those produced using the same DNA vector unless the chromosomal DNA sequence (flanking DNA) adjacent to the inserted transgene is known. For example, Windels et al. use a pair of primers spanning the junction between the inserted transgene and flanking DNA to identify herbicide tolerant soybean event 40-3-2 in 1999, one primer specifically includes the sequence of the inserted transgene and the other primer includes the sequence of the flanking DNA (U.S. Pat. Nos. 6,893,826; 6,825,400; 6,740,488; 6,733,974 and 6,689,880; 6,900,014 and 6,818,807).